Differential control of RNA demethylase activity and selectivity by cofactor ascorbate

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenases (FOGDDs) are crucial enzymes modifying RNA, DNA, and peptides.
• Ascorbate (Vitamin C) is a known cofactor for many FOGDDs, but its specific roles and requirements are not fully understood.
• Understanding ascorbate's interaction with FOGDDs is key to deciphering cellular regulation and disease mechanisms.

Purpose of the Study:

• To investigate the differential ascorbate requirements of FOGDD enzymes, specifically RNA demethylases FTO and AlkBH5.
• To elucidate how ascorbate affects the demethylation of various RNA substrates by FTO.
• To determine the structural basis of ascorbate's interaction with the FOGDD active site.

Main Methods:

• Enzymatic assays were performed using FTO and AlkBH5 with different RNA substrates.
• Biochemical experiments analyzed the decarboxylation of 2-oxoglutarate and substrate oxidation kinetics.
• X-ray crystallography was employed to determine the structure of FTO in complex with ascorbate.

Main Results:

• FTO and AlkBH5 exhibit distinct ascorbate dependencies for RNA demethylation.
• FTO shows variable ascorbate requirements depending on the specific methylated RNA substrate.
• Enzyme kinetics and a crystal structure reveal how ascorbate selectively engages the FOGDD active site.

Conclusions:

• Ascorbate's role in FOGDD catalysis is highly variable, influenced by enzyme-specific factors and substrate interactions.
• The findings provide a structural basis for ascorbate's selective tuning of FOGDD activity.
• Cellular ascorbate levels may dynamically regulate FOGDD-catalyzed reactions, with implications for various cellular processes and diseases.